Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker's Respiratory Allergy.

Wheat allergens are responsible for symptoms in 60-70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21-27 kDa that were recognized by the pooled baker's IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.

Molecular and biochemical characterization of key enzymes in the cysteine and serine metabolic pathways of Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. METHODS: The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. RESULTS: The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. CONCLUSIONS: This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.

[Preparation and identification of anti-human epididymal sperm protein P34H monoclonal antibody].

OBJECTIVE: To prepare and identify anti-human epididymal sperm protein P34H monoclonal antibody (McAb). METHODS: The previously purified recombinant P34H protein was used as an antigen to immunize BALB/c mouse. Cell strains secreting anti-P34H McAb were established by hybridoma technique and then ascitic fluid-type McAb prepared. The sensitivity and specificity of McAb were detected by ELISA and Western blot, respectively. RESULTS: One strain (2C4) of IgG1 Kappa anti-P34H McAb was harvested. The titer detected by ELISA technique was 1:10(3) - 1:10(5). Western blot of healthy human sperm samples and purified recombinant P34H antigen probed with the prepared McAb were immunoreactive at 34,000 and 27,000, respectively. CONCLUSION: Anti-P34H McAb has been prepared successfully by the above methods, which may provide a powerful tool for exploring the relationship between P34H and male fertility.

[Preparation and characterization of monoclonal antibody against DCXR].

AIM: To prepare monoclonal antibodies (mAbs) against Dicarbonyl/L-xylulose reductase (DCXR). METHODS: Normal human liver tissues were homogenized, and mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were detected by ELISA, Western blot and immunohistochemistry. The antibody specificities were identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening. RESULTS: One clone of hybridoma AFF091 secreting specific mAb against DCXR was obtained. The Ig subclass of the mAb was IgG1 and it can be used in ELISA, Western blot, immunohistochemistry, and immunoprecipitation. CONCLUSION: A hybridoma cell line stably secreting specific mAb against DCXR was established. The specific mAb against DCXR would be very useful for investigation of DCXR functions and distribution.

Production of polyclonal antibodies in rabbits is simplified using perforated plastic golf balls.

Production of polyclonal antibodies in the lumen of a perforated golf ball implanted surgically under the skin of a rabbit offers advantages over conventional techniques. Less stress is placed on the rabbit because bleeding is eliminated, complete adjuvants are not used and animal handling is minimized. The technique also offers the advantage that large amounts of antibody-containing fluid can be removed easily from the ball. In this report we describe the surgical protocol and demonstrate use of this technique to produce high-titered antibodies to plant and plant viral proteins.

L-gulono-gamma-lactone oxidase deficiency in rats with osteogenic disorder: enzymological and immunochemical studies.

A strain of Wistar rats with L-gulono-gamma-lactone oxidase deficiency has recently been established. The activity of L-gulono-gamma-lactone oxidase in hepatic microsomes of the mutant rats was not detectable, while that of heterozygous rats was about half that of normal rats. These results were confirmed by immunological quantitation using antibody directed against this enzyme. Thus, it appears that these mutant rats either possess no enzyme at all or a very aberrant form of the enzyme in the liver. Aldonolactonase, another enzyme participating in the biosynthesis of L-ascorbic acid, was found to be present in normal amount in the mutant strain.

In vitro synthesis of L-gulono-gamma-lactone oxidase by rabbit reticulocyte lysate.

In vitro synthesis of L-gulono-gamma-lactone oxidase [L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8], one of the microsomal flavin enzymes, was performed with poly(A)+ RNA of rat liver using the rabbit reticulocyte lysate system in order to study the biosynthesis of the enzyme. The apparent molecular weight of the synthesized enzyme protein was almost the same as that of the purified L-gulono-gamma-lactone oxidase from rat liver. It was demonstrated that the enzyme protein was not detectable when guinea pig poly(A)+ RNA was used for the translation, indicating that the mRNA for the enzyme is absent in the guinea pig or, if it exists, is not translatable.

Purification and characterization of ribitol-5-phosphate and xylitol-5-phosphate dehydrogenases from strains of Lactobacillus casei.

A simple three-step procedure is described which yields electrophoretically homogeneous preparations of ribitol-5-phosphate dehydrogenase and xylitol-5-phosphate dehydrogenase. The former enzyme is a 115,000-molecular-weight protein composed of two subunits of identical size and is specific for its substrate, ribitol. The xylitol-5-phosphate dehydrogenase exists as a tetrameric protein with a molecular weight of 180,000; this enzyme oxidizes the phosphate esters of both xylitol and D-arabitol. Characterization of the physical, kinetic, and immunological properties of the two enzymes suggests that the functionally similar enzymes may not be structurally related.

Phylogenetic conservation of epitopes in mammalian aldose reductase: application to immunoquantitation.

Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific by Western blot analysis of kidney homogenates. In addition, the antiserum (alpha-BKALR2) reacts with a single electrophoretic species in homogenates from rabbit, porcine, and human kidney. ALR2 has been detected in homogenates of bovine kidney, heart, brain and lens, and estimation of the enzyme level in these tissues was accomplished by densitometric analysis of Western blots. Standard curves using highly purified bovine kidney ALR2 were linear in the range of 5-100 ng; a similar sensitivity was seen in tissue homogenates. The results presented here for the ALR2 level in bovine tissues (kidney greater than heart greater than brain greater than lens) are in agreement with literature values for those tissues from which the enzyme has previously been purified. The interspecies similarity in electrophoretic mobility and the retention of antibody reactivity suggest extensive phylogenetic epitope conservation in mammalian aldose reductase.

Intravascularly administered crosslinked immunoprecipitates of gulonolactone oxidase are toxic and rapidly cleared from the circulation.

Glutaraldehyde crosslinked, immunoprecipitated gulonolactone oxidase, injected intraperitoneally, has significant catalytic activity and is capable of providing long-term therapeutic benefit for the enzyme deficiency disease scurvy. The enzyme is tolerated even in repetitive doses. In the present study, however, we have found that when administered intra-arterially this modified enzyme is quite toxic even in single doses. Prior to administration the enzyme complex was filtered through a 5-microns filter. When administered intravascularly the enzyme is not nearly as active catalytically. In spite of this, activity can be detected in vivo as an elevation of plasma ascorbic acid and prolonged survival of guinea pigs fed without the vitamin. Following administration both activity and the enzyme complex are rapidly removed from the circulation. Liver and spleen are largely responsible for this uptake. Because of its toxicity intra-arterial injection of this form of the enzyme does not appear suitable for enzyme therapy.

Immunochemical characterization of aldo-keto reductases from human tissues.

Aldose reductase, aldehyde reductase and carbonyl reductase constitute a family of monomeric NADPH-dependent oxidoreductases with similar physical and chemical properties. Characterization of the enzymes from human tissues by immunotitration and an enzyme immunoassay indicated that, despite their apparent likeness, the three reductases do not cross-react immunochemically.

Purification and properties of a NAD-linked 1,2-propanediol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244.

NAD-dependent 1,2-propanediol dehydrogenase (EC 1.1.1.4) activity was detected in cell-free crude extracts of various propane-grown bacteria. The enzyme activity was much lower in 1-propanol-grown cells than in propane-grown cells of Pseudomonas fluorescens NRRL B-1244, indicating that the enzyme may be inducible by metabolites of propane subterminal oxidation. 1,2-Propanediol dehydrogenase was purified from propane-grown Ps. fluorescens NRRL B-1244. The purified enzyme fraction shows a single-protein band upon acrylamide gel electrophoresis and has a molecular weight of 760,000. It consists of 10 subunits of identical molecular weight (77,600). It oxidizes diols that possess either two adjacent hydroxy groups, or a hydroxy group with an adjacent carbonyl group. Primary and secondary alcohols are not oxidized. The pH and temperature optima for 1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propanediol and NAD are 2 X 10(-2) and 9 X 10(-5) M, respectively. The 1,2-propanediol dehydrogenase activity was inhibited by strong thiol reagents, but not by metal-chelating agents. The amino acid composition of the purified enzyme was determined. Antisera prepared against purified 1,2-propanediol dehydrogenase from propane-grown Ps. fluorescens NRRL B-1244 formed homologous precipitin bands with isofunctional enzymes derived from propane-grown Arthrobacter sp. NRRL B-11315, Nocardia paraffinica ATCC 21198, and Mycobacterium sp. P2y, but not from propane-grown Pseudomonas multivorans ATCC 17616 and Brevibacterium sp. ATCC 14649, or 1-propanol-grown Ps. fluorescens NRRL B-1244. Isofunctional enzymes derived from methane-grown methylotrophs also showed different immunological and catalytic properties.

Immunochemical properties of NAD+-linked glycerol dehydrogenases from Escherichia coli and Klebsiella pneumoniae.

An NAD+-linked glycerol dehydrogenase hyperproduced by a mutant of Escherichia coli K-12 was found to be immunochemically homologous to a minor glycerol dehydrogenase of unknown physiological function in Klebsiella pneumoniae 1033, but not to the glycerol dehydrogenase of the dha system responsible for anaerobic dissimilation of glycerol or to the 2,3-butanediol dehydrogenase of K. pneumoniae.

Feasibility of using an isolated intestinal segment as an artificial organ for enzyme replacement therapy.

Guinea pigs fed an ascorbic acid-deficient diet develop scurvy because of the absence of the enzyme L-gulonolactone oxidase. In theory if this enzyme is provided and its substrate L-gulonolactone is present at adequate concentrations ascorbic acid will be synthesized and the development of scurvy prevented. Using this model we tested whether a viable segment of intestine could be used to contain the administered enzyme and act as an artificial organ for the production of ascorbic acid. A surgical procedure was developed to prepare an externalized pouch of intestine with its circulation left intact. When enzyme is inserted in this intestinal bag it is not toxic and not antigenic in some animals, whereas, enzyme injected intraperitoneally is clearly antigenic. Synthesis of ascorbic acid by this artificial organ could not, however, be detected by elevation of plasma concentrations of the vitamin.

L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats.

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-gamma-lactone: oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of Fad.